The present invention relates generally to nucleic acids and more specifically to nucleic acid molecules that can be used as probes for detecting and amplifying parvovirus.
The parvoviruses constitute a family of viruses that have been associated with diseases or conditions in multiple mammals, including dogs and humans. Parvovirus strain B19 is associated with diseases and syndromes in humans. Parvovirus strain B19 is an iscoahedral, non-enveloped DNA virus whose genome includes a single-stranded 5.6 kb DNA molecule that encodes two structural proteins, which are designated VP1 and VP2. Also encoded in the parvoviral genome is a non-structural protein, designated NS-1, which is considered important for viral DNA replication.
Several diseases and syndromes associated with parvovirus strain B19, have been described. One disease includes ertyhema infectiosum (EI), which is a common in children and is characterized by fever, headache, nausea, and diarrhea. While these symptoms are typically mild, the consequences of parvovirus infection in some individuals, such as pregnant women, can be more severe. For example, parvovirus B19 infection during pregnancy can have significant and potentially fatal effects on the fetus.
The invention is based in part on the discovery of oligonucleotides that can be used to detect and amplify human parvovirus B19 in a biological sample. The nucleic acids of the invention are referred to herein as PRVX nucleic acids. PRVX nucleic acids of the invention can be used to detect parvovirus in biological samples, such as those containing blood and blood-derived products.
Accordingly, in one aspect the invention features nucleic acids that specifically hybridize to regions of human B19 parvovirus encoding NS and VP. Nucleic acids of the invention include PRV1, PRV2, and PRV3 nucleic acids. Examples of PRV1 and PRV2 nucleic acids of the invention include the primers VINS-3F and VINS-3R, respectively. These primers can be used to amplify a 106 nucleotide sequence from a region encoding a non structural (xe2x80x9cNSxe2x80x9d) protein of a human B19 parvovirus. In some embodiments, amplification occurs in a polymerase chain reaction (xe2x80x9cPCRxe2x80x9d). The VINS-3F oligonucleotide includes the nucleotide sequence 5xe2x80x2-ATGGAGCTATTTAGAGGGGT-3xe2x80x2 (SEQ ID NO:1). The VINS-3R oligonucleotide primer includes the sequence 5xe2x80x2-GTTCCCAGTCAGAAGTGTCT-3xe2x80x2(SEQ ID NO:2). These sequences are highly conserved among human parvovirus strain B19 isolates.
The PCR product generated using VINS-3F and VINS-3 can be detected using a PRV3 nucleic acid of the invention. The PRV3 nucleic acid is based on the nucleotide sequence 5xe2x80x2-TAATGTTCTGGACTGTGCTAACGAT-3xe2x80x2 (SEQ ID NO:3), which recognizes a nucleotide sequence in the region amplified by the VINS-3F and VINS-3R primers.
Also within the invention are PRV4, PRV5, and PRV6 nucleic acids. These nucleic acids are based on oligonucleotides derived from a region of the human B19 parvovirus encoding the viral protein (VP). PRV4 and PRV5 nucleic acids include VIVP-1F and VIVP-1R, respectively. A VIVP-IF nucleic acid includes the sequence 5xe2x80x2-CCCAGAGCACCATTATAAGGTGTT-3xe2x80x2(SEQ ID NO:4), which is highly conserved among parvovirus B19 isolates. A VIVP-IR nucleic acid includes the sequence 5xe2x80x2-TTATGGGACTAATGGTGCAAACC-3xe2x80x2 (SEQ ID NO:5), which is also highly conserved among human parvovirus B19 isolates. The VIVP-1F and VIVP-1R primer pair can specifically amplify a 95 nucleotide sequence of the human parvovirus B-19 VP region. This is shown schematically in FIG. 1.
The identity of an amplification product obtained using VIVP-1F and VIVP-1R can be confirmed by hybridizing the PCR product to a PRV6 nucleic acid of the invention. The PRV6 nucleic acid of the invention is based on an oligonucleotide named VIVP-1P, which recognizes nucleotides found between the sequences amplified by the VIVP-1F and VIVP-1R primers. The VIVP-IP probe includes the sequence 5xe2x80x2-CACAATGCCAGTGGAAAGGAGGCAA-3xe2x80x2 (SEQ ID NO:6), which is highly conserved among human parvovirus B19 strains.
Also within the invention are PRV7, PRV8, PRV9, and PRV10 nucleic acid sequences. These nucleic acids hybridize to regions encoding a viral polypeptide (xe2x80x9cVPxe2x80x9d) of a porcine parvovirus genome. These sequences are based on the primers VIPPVP-1F, VIPPVP-1R, VIPPVP-1P, and VIPPVP-1F(Alt), respectively. The VIPPVP-1F primer includes the sequence 5xe2x80x2-GACCAAGGAGAACCAACTAA-3xe2x80x2 (SEQ ID NO:7), while the VIPPVP-1R primer includes the sequence 5xe2x80x2-TTCATCAGCTGCTGAGAAGT-3xe2x80x2 (SEQ ID NO:8). The VIPPVP-1P nucleic acid sequence includes the sequence 5xe2x80x2-AAAAGAACACGACGAAGCCTACGACAAATA-3xe2x80x2 (SEQ ID NO:9), and the VIPPVP F(Alt) primer includes the sequence 5xe2x80x2-ACAGGACTAACTCTACCAGG-3xe2x80x2 (SE IQ NO:10). The VIPPVP-1R primer and either the VIPPVVP-1F or VIPPVP-1F(Alt) primer can be used to amplify a region of a porcine parvovirus encoding a VP protein. The VIPPVP-1P nucleic acid hybridizes to nucleic acids located within the sequence amplified by the VIPPVP-1R primer and either the VIPPVP-1F or VIPPVP-1F(Alt) primer. Accordingly, it can be used to confirm the identity of these sequences.
In addition to detecting the presence of a parvovirus in a sample of nucleic acids, these sequences can be used to amplify a control porcine parvovirus added to a sample suspected of containing a human parvovirus sample. In one embodiment of the invention, a PRV8 (e.g., SEQ ID NO:8) and a PRV7 or PRV10 (e.g., SEQ ID NO:7 or SEQ ID NO:10 nucleic acid is added along with a porcine parvovirus to a sample suspected of containing human parvovirus B19 strain, and a primer pair including PRV1 and PRV2 (e.g., SEQ ID NO:1 and SEQ ID NO:2) or PRV4 and PRV5 (e.g., SEQ ID NO:4 or SEQ ID NO:5), or both primer pairs, and amplification products are identified. The identity of amplification products can be confirmed using the corresponding confirmatory probe for a given primer pair, e.g. PRV10 (e.g., SEQ ID NO:10) when PRV7 and PRV8 nucleic acids are used as primers, PRV3 (e.g., SEQ ID NO:3) when PRV1 and PRV2 are used as primers, or PRV6 (e.g., SEQ ID NO:6)when PRV4 and PRV5 are used as primers.
Also included in the invention are reaction systems that include a target nucleic acid, a PRV nucleic acid system, and a polymerase. The PRVX nucleic acids are preferably present in pairs to allow for PCR mediated amplification of parvovirus in a target sample. Also provided by the invention are kits containing one or more PRVX nucleic acids.
The invention also provides methods for identifying a parvovirus nucleic acid in a sample by contacting a sample containing, or suspected of containing, a parvovirus nucleic acid with a PRVX nucleic acid. In some embodiments, the methods include PCR-mediated amplification of the parvoviral nucleic acid using one or more of the PRV primer pairs described herein. The methods may be used with various biological samples including, e.g., blood and/or blood-derived compositions (e.g., clotting factors, plasma, serum).
In additional aspects, the invention includes a method of amplifying a parvovirus nucleic acid present in a target sample, as well as methods of diagnosing diseases or conditions associated with parvovirus infection.
The PRV1, PRV2, PRV3, PRV4, PRV5 and PRV6 nucleic acids hybridize to most known parvovirus B19 strains and allow for the PCR-mediated amplification of the vast majority of parvovirus B19 strains. Also included in the invention is the use of a second parvovirus and PRV nucleic acids that specifically detect the second parvovirus. The second parvovirus, which is preferably porcine parvovirus, can be added to a human nucleic acid sample and used as an internal control to facilitate the monitoring of the overall efficiency of nucleic acid extraction and the presence of potentially interfering substances within the PCR amplification reaction. The porcine parvovirus is introduced into the samples and this parvoviral DNA is co-purified and co-amplified with the B19 DNA.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.